Cancer accounts for over one-fifth of the total mortality in the United States, making it the second leading cause of death. The leading types of cancer are lung, prostate (men), colorectal, and breast (women). Currently, most cancers are treated by surgery, chemotherapy, radiation therapy, or a combination of the above.
A disadvantage of chemotherapy and radiation therapy is that, in addition to destroying the cancer, they also destroy or suppress the main blood-forming organ in the body, the bone marrow, resulting in reduced blood-clotting and infection-fighting capability in the patient. The phenomena of marrow damage or destruction are referred to, respectively, as myelosuppression or myeloablation.
Hematopoietic cells arise in the bone marrow from a totipotent stem cell which is characterized as being able to renew itself as well as to give rise to cells of all the other hematopoietic lineages, including the erythroid, megakaryocytic, myeloid, and lymphoid lineages. This stem cell gives rise to progenitor cells, for example, erythroid progenitors and myeloid progenitors, which are committed to differentiate along a specific lineage. Progenitor cells, in turn, give rise to differentiated cells which are morphologically recognizable as belonging to a certain lineage and which have a limited or no capacity to proliferate. In humans, stem cells and progenitor cells express the CD34 antigen, while more differentiated hematopoietic cells do not.
To avoid the above-mentioned problem of myelosuppression, cancer therapy is often given in divided doses, which allows the bone marrow to recover between treatment cycles. Unfortunately, this method of treatment allows the cancer to grow back and to develop resistance to the chemotherapeutic agents being used to treat it. Use of higher doses of chemotherapy or radiation therapy is expected to result in higher cure rates among cancer patients, but may destroy the bone marrow completely (myeloablation), necessitating that the patient be transplanted with bone marrow from a histocompatible donor (an allogeneic transplant) or with his own marrow (an autologous transplant), harvested prior to myeloablative treatment.
The ability to culture hematopoietic cells and their precursors, derived from the bone marrow, peripheral blood, or umbilical cord blood of the patient or a suitable donor, offers the potential to overcome the disadvantages of high-dose cancer therapy by providing an expanding source of cells which can be used to reconstitute the patient's blood-clotting and infection-fighting functions between cycles of therapy. In addition, the ability to expand hematopoietic cells and their precursors in vitro may relieve dependence on bone marrow aspiration or multiple aphereses as the only means of obtaining sufficient cells for transplantation.
It is generally believed in the art that in vitro growth of hematopoietic cells and their precursors requires both the appropriate growth factors (cytokines), or combination thereof, and the presence of bone marrow stromal cells. The latter are believed to be particularly important and are thought to function both as sites of attachment for developing hematopoietic cells and as sources of as yet unidentified growth factors required by the cells for proliferation and differentiation. In the absence of stromal elements, it is believed that hematopoietic precursor cells do not expand in number.
Early work in the field of hematopoietic stem cell culture centered around the culture of bone marrow aspirates from mouse in agar gel or liquid medium. Unfractionated bone marrow (including stem cells, progenitor cells, more differentiated hematopoietic cells, and stromal elements) was used to inoculate these cultures, which were generally short-lived and resulted in little or no increase in cell number, particularly in the stem cell and progenitor compartments. The results were even less promising when human bone marrow was employed. The cells were generally observed to adhere to the bottom and sides of the vessel in which they were cultured and their removal therefrom was difficult.
Subsequent efforts focused on inoculating whole bone marrow, again mouse, onto pre-established monolayers of bone marrow stromal cells (so-called Dexter cultures; Dexter, Acta Haematol. 62: 299-305, 1979). While some success was obtained in the mouse with Dexter cultures, the same approach was disappointing in the human. Various investigators have observed a steady decline in the numbers of all cell types in human Dexter cultures (reviewed in Quesenberry, Curr. Topics in Microbiol. Immunol. 177: 151, 1992).
A further disadvantage of Dexter cultures is that, to the extent that there is expansion of hematopoietic precursor cells, these cells adhere to the stromal layer and are extremely difficult to recover from the culture without employing conditions which damage the cells. The proliferating cells which are released into the culture medium (that is, the non-adherent cells) are generally more mature cells, which cannot restore sustained hematopoiesis in a transplanted individual.
Several investigators have attempted to improve upon the Dexter culture. For example, Naughton et al. describe the establishment of the stromal cell layer on a nylon screen (Bone Marrow Purging and Processing, Alan R. Liss (pub.), pg. 435-445, 1990). Emerson et al. have used a protein matrix, such as collagen-coated beads or membranes, to support the stromal cell layer (WO90/15877, published Dec. 27, 1990 and WO92/11355, published Jul. 9, 1992). Neither of these methods, however, overcomes the disadvantage mentioned above, namely, that hematopoietic precursor cells adhere to the stromal layer, from which they are not easily removed.
There remains a need in the art for a method of culturing human hematopoietic cells, which method (a)is independent of the presence of bone marrow stromal elements; (b) results in expansion of the number of hematopoietic precursor cells; and (c) enhances the yield and recovery of the precursor cells without compromising viability. The present invention fulfills this need and further provides other related advantages.